EP3318630B1 - Méthode de tri de cellules tissulaires - Google Patents

Méthode de tri de cellules tissulaires Download PDF

Info

Publication number
EP3318630B1
EP3318630B1 EP16783256.7A EP16783256A EP3318630B1 EP 3318630 B1 EP3318630 B1 EP 3318630B1 EP 16783256 A EP16783256 A EP 16783256A EP 3318630 B1 EP3318630 B1 EP 3318630B1
Authority
EP
European Patent Office
Prior art keywords
cell
cells
mirna
gene
mir
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
EP16783256.7A
Other languages
German (de)
English (en)
Other versions
EP3318630A1 (fr
EP3318630A4 (fr
Inventor
Yoshinori Yoshida
Hirohide Saito
Kenji Miki
Kei Endo
Seiya Takahashi
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
IPS ACADEMIA JAPAN Inc
Original Assignee
Ips Academia Japan Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ips Academia Japan Inc filed Critical Ips Academia Japan Inc
Publication of EP3318630A1 publication Critical patent/EP3318630A1/fr
Publication of EP3318630A4 publication Critical patent/EP3318630A4/fr
Application granted granted Critical
Publication of EP3318630B1 publication Critical patent/EP3318630B1/fr
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/067—Hepatocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0676—Pancreatic cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/069—Vascular Endothelial cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the present invention relates to a method for sorting tissue cells, and in particular, relates to a method for sorting an endothelial cell, a hepatocyte, and an insulin-producing cell.
  • Non-Patent Literatures 1, 2, and 3 Much attention has been paid to cell transplantation therapy using pluripotent stem cells, and methods for inducing each tissue cell have been reported. However, all the cells induced are not just a desired type of cell and usually contain other types of cell at the same time. To use such a cell population as cells used for transplantation, it is necessary to increase the purity of a desired type of cell by sorting, etc.
  • WO 2011/154553 A2 is concerned with the use of human-derived microRNAs as targets for the identification of cardiac and cardiac-like cell types. Summary of Invention Problems to be Solved by the Invention
  • an object of the present invention is to provide a tool for sorting each type of tissue cell.
  • each miRNA-responsive mRNA can be used to allow a type of tissue cell such as an endothelial cell, a hepatocyte, or an insulin-producing cell to be purified with high purity.
  • miRNAs expressed specifically in each tissue cell population were identified by microarray analysis.
  • an mRNA (miRNA switch) having a marker gene and a target sequence of each of the above miRNAs was constructed.
  • this mRNA was introduced into cells that were subject to sorting.
  • cells exhibiting different behavior were found to be present.
  • these cells were confirmed to be a target type of tissue cell with high purity. In this way, a desired tissue cell sorting method was discovered.
  • the present invention has been completed based on such findings.
  • the present invention provides the following items.
  • tissue cells which are endothelial cells, hepatocytes, or insulin-producing cells can be easily sorted and purified to high purity.
  • the mRNA that has been introduced into a cell population using the method according to the present invention is only transiently present in cells. Therefore, this makes it possible to sort target cells safely without the mRNA being introduced into the genome.
  • the present invention discloses a method in which living cells can be sorted, without fixation, on the basis of the level of expression of an intracellular active miRNA. Further, cells purified in accordance with the method of the present invention can be used for transplantation therapy.
  • the miRNA-responsive mRNA is also simply called an miRNA switch, and means an mRNA comprising the following nucleic acid sequences (i) and (ii):
  • the "miRNA” refers to a non-coding RNA composed of a short chain (20 to 25 bases) that is present in a cell and is participated in the regulation of gene expression through mRNA degradation and/or inhibition of translation from an mRNA to a protein.
  • This miRNA is first produced as a transcribed, single-stranded pri-miRNA, an miRNA of which, together with its complementary strand, can form a hairpin loop structure; a portion of the pri-miRNA is cleaved by an enzyme called Drosha present in the nucleus to give a pre-miRNA, which is exported outside the nucleus; and the pre-miRNA is further cleaved by Dicer to become functional.
  • the miRNA of (i) can be appropriately selected from an miRNA specifically expressed in a desired type of cell to be sorted.
  • the desired type of cell is an endothelial cell, a hepatocyte, or an insulin-producing cell, as described below.
  • the miRNA specifically expressed may be miRNAs, the expression levels of which are higher in a desired type of cell than in another type of cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more.
  • Such an miRNA can be suitably selected from miRNAs registered on database information site (e.g., http://www.mirbase.org/ or http://www.microrna.org/) and/or miRNAs described in literature disclosed in the database. Examples include, but are not limited to, miRNAs listed in Table 1.
  • the nucleic acid sequence specifically recognized by an miRNA is preferably a sequence perfectly complementary to the miRNA.
  • the nucleic acid sequence may have a mismatch in the perfectly complementary sequence as long as the nucleic acid sequence can be recognized by the miRNA.
  • the mismatch may be a mismatch that the miRNA can usually recognize.
  • the degree of the mismatch may be about 40 to 50%. Examples of such a mismatch include, but are not particularly limited to, 1 base, 2 bases, 3 bases, 4 bases, 5 bases, 6 bases, 7 bases, 8 bases, 9 bases, and 10 bases; or 1% mismatch, 5% mismatch, 10% mismatch, 20% mismatch, 30% mismatch, and 40% mismatch per entire recognition sequence.
  • mismatches may be included in the 5' region of a target sequence, which region is outside a seed region and corresponds to about 16 bases located on the 3' side of the miRNA.
  • the seed region may contain no mismatch or may contain mismatches: 1 base, 2 bases, or 3 bases.
  • the length of such a sequence may be a base length corresponding to the number of bases that can bind specifically to RISC.
  • the length of the sequence is preferably 18 bases or more and less than 24 bases and more preferably 20 bases or more and less than 22 bases.
  • nucleic acid sequence specifically recognized by an miRNA may be used after determined by introducing an miRNA-responsive mRNA having the sequence into a desired type of cell and other cells; and by verifying that expression of a relevant marker gene is inhibited only in the desired type of cell.
  • Examples of nucleic acid sequences specifically recognized by miR-126-3P, miR-126-5P, miR-122-5P, and miR-375 include, but are not limited to, target sequences listed in Table 2.
  • miRNAs and the target sequences thereof [Table 2] miRNA name miRNA target sequence (5' -> 3') SEQ ID NO: miR-126-3P CGCATTATTACTCACGGTACGA 5 miR-126-5P CGCGTACCAAAAGTAATAATG 6 miR-122-5P CAAACACCATTGTCACACTCCA 7 miR-375 TCACGCGAGCCGAACGAACAAA 8
  • marker gene refers to a gene encoding any protein that functions as a marker after translation in a cell and enables cell sorting.
  • Examples of a gene encoding the protein that functions as a marker after translation in a cell include, but are not limited to, genes encoding fluorescent proteins, light-emitting proteins, chromogenic proteins or fluorescence proteins; genes encoding proteins that help fluorescent proteins, etc., emit fluorescence, light, or color for visualization and quantification; and genes encoding proteins that kill cells when expressed (e.g., a membrane protein-encoding gene, an apoptosis-inducing gene, a suicide gene).
  • an apoptosis-inhibiting gene may be used as another marker gene.
  • a marker protein refers to a protein translated from an mRNA containing a nucleic acid corresponding to a coding region of the marker gene.
  • first is a term used to distinguish between two marker genes when used and is not restricted to a specific marker gene.
  • fluorescent protein examples include, but are not limited to, blue fluorescent proteins (e.g., Sirius, BFP, EBFP); cyan fluorescent proteins (e.g., mTurquoise, TagCFP, AmCyan, mTFP1, MidoriishiCyan, CFP); green fluorescent proteins (e.g., TurboGFP, AcGFP, TagGFP, Azami-Green (e.g., hmAG1), ZsGreen, EmGFP, EGFP, GFP2, HyPer); yellow fluorescent proteins (e.g., TagYFP, EYFP, Venus, YFP, PhiYFP, PhiYFP-m, TurboYFP, ZsYellow, mBanana); orange fluorescent proteins (e.g., KusabiraOrange (e.g., hmK02), mOrange); red fluorescent proteins (e.g., TurboRFP, DsRed-Express, DsRed2, TagRFP, DsRed-Monomer, AsRe
  • examples of the light-emitting protein include, but are not limited to, Aequorin.
  • examples of the protein that helps emit fluorescence, light, or color include, but are not limited to, enzymes that degrade fluorescent, light-emitting, or chromogenic precursors (e.g., luciferase, phosphatase, peroxidase, ⁇ -lactamase).
  • a relevant precursor is made to contact a cell population or the relevant precursor is introduced into the cell population during target cell sorting.
  • the apoptosis-inducing gene means a gene encoding a protein having activity of inducing cellular apoptosis. Examples include, but are not limited to, I ⁇ B, Smac/DIABLO, ICE, HtrA2/OMI, AIF, endonuclease G, Bax, Bak, Noxa, Hrk (harakiri), Mtd, Bim, Bad, Bid, PUMA, activated caspase-3, Fas, and Tk.
  • Bim is preferably used as the apoptosis-inducing gene.
  • a suicide gene is a gene, the cellular expression of which is lethal to the cell.
  • the suicide gene product e.g., diphtheria toxin A
  • the suicide gene product by itself, may cause cell death.
  • expression of such a gene may make cells susceptible to a specific chemical (e.g., expression of herpes simplex virus thymidine kinase gene can make cells susceptible to an anti-virus compound).
  • the suicide gene examples include, but are not limited to, genes encoding: diphtheria toxin A; herpes simplex virus thymidine kinase (HSV-TK); carboxypeptidase G2 (CPG2); carboxyl esterase (CA); cytosine deaminase (CD); cytochrome P450 (cyt-450); deoxycytidine kinase (dCK); nitroreductase (NR); purine-nucleoside phosphorylase (PNP); thymidine phosphorylase (TP); varicella-zoster virus thymidine kinase (VZV-TK); and xanthine-guanine phosphoribosyl transferase (XGPRT).
  • HSV-TK gene is preferably used as the suicide gene.
  • the marker gene may include a gene encoding a localization signal.
  • the localization signal include a nuclear localization signal, a plasma membrane localization signal, a mitochondrial localization signal, and a protein secretion signal. Specific examples include, but are not limited to, a classical nuclear localization signal (NLS), M9 sequence, a mitochondrial target sequence (MTS), and an ER localization sequence.
  • NLS classical nuclear localization signal
  • M9 M9 sequence
  • MTS mitochondrial target sequence
  • ER localization sequence an ER localization sequence.
  • a nucleic acid sequence specifically recognized by an miRNA is operably linked to a nucleic acid sequence corresponding to a coding region of a first marker gene
  • a nucleic acid sequence specifically recognized by an miRNA is operably linked to a nucleic acid sequence corresponding to a coding region of a first marker gene
  • at least one miRNA target sequence is included in a 5' UTR, a 3' UTR of an open reading frame (including a start codon) encoding a marker gene and/or the open reading frame.
  • An miRNA-responsive mRNA includes, preferably from the 5' end, in the 5' to 3' direction, a cap structure (7-methylguanosine 5'-phosphate), and an open reading frame encoding a marker gene, and a poly-A tail, and also includes at least one miRNA target sequence in a 5' UTR, a 3' UTR, and/or the open reading frame.
  • the position of the miRNA target sequence of the mRNA may be located at the 5' UTR or 3' UTR or located within the open reading frame (on the 3' side of the start codon), all of which may each be provided with an miRNA target sequence.
  • the number of the miRNA target sequences may be 1, 2, 3, 4, 5, 6, 7, 8, or more.
  • the nucleic acid sequences (i) and (ii) of the miRNA-responsive mRNA are linked from the 5' to 3' direction in this order.
  • the number and kinds of bases between the cap structure and the miRNA target sequence may be freely chosen as long as the bases involve neither a stem structure nor a specific conformation.
  • the number of bases between the cap structure and the miRNA target sequence is from 0 to 50 and preferably from 10 to 30.
  • the number and kinds of bases between the miRNA target sequence and the start codon may be freely chosen as long as the bases involve neither a stem structure nor a specific conformation.
  • the number of bases between the miRNA target sequence and the start codon is from 0 to 50 and preferably from 10 to 30.
  • the miRNA target sequence of an miRNA-responsive mRNA preferably includes no AUG, a start codon.
  • the AUG is in the same frame of a marker gene linked on the 3' side.
  • the AUG in the target sequence may be changed to GUG.
  • the position of the target sequence in the 5' UTR may be changed appropriately. For instance, in some position designs, the number of bases between the cap structure and the AUG sequence in the target sequence is from 0 to 60 such as from 0 to 15, from 10 to 20, from 20 to 30, from 30 to 40, from 40 to 50, and from 50 to 60.
  • an miRNA target sequence is present in a 5' UTR in the present invention
  • the following sequences for example, may be used.
  • miRNAs and the 5' UTR sequences of corresponding miRNA-responsive mRNAs [Table 3] miRNA name 5' UTR sequence (5' -> 3') in miRNA-responsive mRNA (AUG at the 3' end indicates a start codon) SEQ ID NO: miR-126-3P 9 miR-126-5P 10 miR-122-5P 11 miR-375 12
  • the following sequence may be used downstream (i.e., in a 3' UTR) of a subsequent marker gene.
  • any one of the above-described genes may be used as the marker gene interposed between the 5' UTR and the 3' UTR.
  • a gene encoding BFP for example, is used as a marker gene in the present invention
  • the following sequences may each be employed as a full-length miRNA-responsive mRNA according to the present invention.
  • an miRNA-responsive mRNA can be introduced as a form of mRNA.
  • RNA-responsive mRNA is the form of RNA
  • examples of a technique for introducing the mRNA into a somatic cell include lipofection and microinjection.
  • 5-methylcytidine and/or pseudouridine may be incorporated into the RNA to be used ( Warren L, (2010) Cell Stem Cell. 7: 618-630 ).
  • modified bases all or some of uridines or cytidines may be replaced by the modified base independently. When some of them are replaced, the percentage of the modified base may be freely chosen and the position or positions may be at random.
  • Two or more different miRNA-responsive mRNAs may be introduced.
  • an miRNA-responsive mRNA and an mRNA as a control hereinafter, sometimes referred to as a control mRNA
  • multiple mRNAs are preferably co-introduced.
  • the ratio between two or more co-introduced mRNAs can be maintained in individual cells. So, the activity ratio between proteins expressed using these mRNAs can be also maintained in a corresponding cell population.
  • the amount of each mRNA introduced varies depending on a cell population into which the mRNAs are introduced, the mRNAs introduced, and a method and reagent for the introduction, and may be suitably selected by those skilled in the art so as to achieve a desired level of translation thereof.
  • a control mRNA for example, consists of a sequence comprising a nucleic acid corresponding to a coding region of a marker gene or a sequence comprising a nucleic acid corresponding to a coding region of a drug resistance gene, and does not have an miRNA target site.
  • the control mRNA together with an miRNA-responsive mRNA is introduced into a cell population. Then, the control mRNA can function as a control for identifying and distinguishing a cell containing the miRNA-responsive mRNA from others.
  • control mRNA can also function as a control used when a level of signal (e.g., fluorescence and light) from the miRNA-responsive mRNA is quantified.
  • a level of signal e.g., fluorescence and light
  • the amount of the control mRNA introduced may be suitably selected by those skilled in the art so as to achieve a desired level of translation thereof.
  • drug resistance gene it is possible to use any gene that can express a protein resistant to a corresponding drug.
  • examples include, but are not limited to, antibiotic resistance genes.
  • the antibiotic resistance gene include a kanamycin resistance gene, an ampicillin resistance gene, a puromycin resistance gene, a blasticidin resistance gene, a gentamicin resistance gene, a kanamycin resistance gene, a tetracycline resistance gene, and a chloramphenicol resistance gene.
  • a puromycin resistance gene or a blasticidin resistance gene is preferably used as the antibiotic resistance gene.
  • a sorting method more preferably includes a step of simultaneously introducing an miRNA-responsive mRNA and a control mRNA into a cell population containing a target cell.
  • the step may be preferably performed by co-transfecting the miRNA-responsive mRNA and the control mRNA.
  • Use of the control mRNA allows for sorting of, as a target cell, a cell in which a marker protein is less or hardly translated from the miRNA-responsive mRNA even if the efficiency of introducing the miRNA-responsive mRNA into cells is low.
  • a marker gene included in the control mRNA is preferably different from a marker gene included in an miRNA-responsive mRNA.
  • the marker gene included in an miRNA-responsive mRNA is an apoptosis-inducing gene or a suicide gene
  • the marker gene included in the control mRNA may be a fluorescent protein-encoding gene.
  • fluorescent cells are subjected to, for example, FACS sorting for selective separation. Then, cells with reduced levels of expression of the apoptosis-inducing gene or the suicide gene are sorted as target cells. This can improve sorting precision.
  • a control mRNA consisting of a sequence comprising a nucleic acid corresponding to a coding region of a drug resistance gene can be used with an miRNA-responsive mRNA consisting of a sequence comprising a nucleic acid corresponding to a coding region of any marker gene.
  • miRNA-responsive mRNA consisting of a sequence comprising a nucleic acid corresponding to a coding region of any marker gene.
  • cells containing the miRNA-responsive mRNA are resistant to the drug regardless of the kind of the marker gene, which can improve sorting precision.
  • the same type of gene may be used for marker genes included in an miRNA-responsive mRNA and a control mRNA.
  • fluorescent protein-encoding genes may be used for the marker genes included in an miRNA-responsive mRNA and a control mRNA. In this case, two corresponding fluorescent proteins should have different fluorescence wavelengths.
  • the fluorescent protein-encoding gene may be replaced by a gene encoding a light-emitting protein or a protein that helps emit fluorescence, light, or color, which gene is likewise used in combination.
  • control mRNA examples include, but are not particularly limited to, the following mRNAs.
  • a sorting method may comprise a step of introducing an miRNA-responsive mRNA and an optional control mRNA into a cell population.
  • This step allows a target type of cell to be sortable from the cell population containing the target type of cell.
  • this step enables a desired type of cell to provide detectable signal information different from signal information of the other types of cell in the cell population containing two or more types of cell optionally including the target type of cell.
  • the sorting step may further be carried out to selectively separate the target type of cell.
  • endothelial cells are squamous thin cells constituting the inner surface of a blood vessel and are not distinguished from endothelial precursor cells in the present invention.
  • Endothelial cells used in the present invention may be continuously cultured to form a tubular structure.
  • the cells can take up acetylated low-density-lipoproteins (ac-LDL).
  • ac-LDL acetylated low-density-lipoproteins
  • the endothelial cells are characterized by expression of a marker including, without particular limitation, CD31, etc.
  • a nucleic acid sequence that is specifically recognized by an miRNA and is contained in an miRNA-responsive mRNA used for sorting an endothelial cell is preferably a nucleic acid sequence specifically recognized by miR-126-3P or miR-126-SP.
  • a cell population that is subject to sorting may contain any cell population that can contain endothelial cells.
  • the cell population may be a cell population that has been induced to differentiate from a pluripotent stem cell or may be an in vivo cell population, but is not limited thereto.
  • an miRNA-responsive mRNA is introduced into a cell population that may or may not contain endothelial cells.
  • the cell population may be a cell population that has been induced to differentiate from a pluripotent stem cell.
  • Such induced differentiation is not limited to specific induction into endothelial cells, and may include induced differentiation for the purpose of obtaining another type of cell such as cardiomyocytes.
  • a protocol for producing endothelial cells from a pluripotent stem cell may be appropriately performed, including, for example, the known procedure ( Li et al., J. Cell Biochem., 106: 194-199, 2009 ).
  • hepatocytes are defined as cells expressing an mRNA of albumin or HNF4A. According to another embodiment of the present invention, hepatocytes are defined as cells characterized by any one or combination of known hepatocyte functions such as glycogen accumulation, low-density-lipoprotein (LDL) incorporation, albumin secretion, ammonia metabolism and urea synthesis, cytochrome P450 activity, lipid metabolism, and drug metabolism.
  • LDL low-density-lipoprotein
  • a nucleic acid sequence that is specifically recognized by an miRNA and is contained in an miRNA-responsive mRNA used for sorting a hepatocyte is preferably a nucleic acid sequence specifically recognized by miR-122-SP.
  • a cell population that is subject to sorting may contain any cell population that can contain hepatocytes.
  • the cell population may be a cell population that has been induced to differentiate from a pluripotent stem cell or may be an in vivo cell population, but is not limited thereto.
  • an miRNA-responsive mRNA is introduced into a cell population that may or may not contain hepatocytes.
  • the cell population may be a cell population that has been induced to differentiate from a pluripotent stem cell. Such induced differentiation is not limited to specific induction into hepatocytes.
  • a protocol for producing hepatocytes from a pluripotent stem cell may be appropriately performed, including the known procedures (e.g., WO2001/081549 ; Takayama K, et al., Stem Cell Reports. 1: 322-335, 2013 ; Kajiwara M, et al., Proc Natl Acad Sci U S A. 109: 12538-12543, 2012 ; Hay DC, et al., Stem Cells. 26: 894-902, 2008 ).
  • insulin-producing cells are defined as cells expressing an mRNA of insulin.
  • a nucleic acid sequence that is specifically recognized by an miRNA and is contained in an miRNA-responsive mRNA used for sorting an insulin-producing cell is preferably a nucleic acid sequence specifically recognized by miR-375.
  • a cell population that is subject to sorting may contain any cell population that can contain insulin-producing cells.
  • the cell population may be a cell population that has been induced to differentiate from a pluripotent stem cell or may be an in vivo cell population, but is not limited thereto.
  • an miRNA-responsive mRNA it may be possible for an miRNA-responsive mRNA to be introduced into a cell population that may or may not contain insulin-producing cells.
  • the cell population may be a cell population that has been induced to differentiate from a pluripotent stem cell. Such induced differentiation is not limited to specific induction into insulin-producing cells.
  • Examples of a protocol for inducing insulin-producing cells from a pluripotent stem cell include methods for induced differentiation using activin and/or retinoic acid (RA) (Japanese Patent Laid-Open No. 2009-225661 , E. Kroon et al., Nature Biotechnology (2008) Vol. 26, No. 4: 443-452 , K. A. D'Amour et al., Nature Biotechnology (2006) Vol. 24, No. 11: 1392-1401 , W. Jiang, Cell Research (2007) 17: 333-344, J . H. Shim et al., Diabetologia (2007) 50: 1228-1238 , R. Maehra et al., PNAS (2009), vol. 106, No.
  • RA retinoic acid
  • Additional examples include: methods including introducing PDX1 into a pluripotent stem cell and culturing the cell ( US Patent No. 7,534,608 and Japanese Patent Laid-Open No. 2006-075022 ); and methods for producing insulin-producing cells by applying a suitable combination of low-molecular-weight compounds to a pluripotent stem cell ( WO2011/081222 and Kunisada Y et al., Stem Cell Res. (2012) vol. 8, No. 2: 274-284 ).
  • a pluripotent stem cell is a stem cell that is pluripotent and can differentiate into many types of cells in vivo and can also self-replicate, and includes any type of cell that can be induced into hepatocytes used in the present invention.
  • the pluripotent stem cell include, but are not particularly limited to, an embryonic stem (ES) cell, a nuclear transfer-mediated clone embryo-derived embryonic stem (ntES) cell, a germline stem cell (“GS cell”), an embryonic germ cell (“EG cell”), an induced pluripotent stem (iPS) cell, and a fibroblast or bone marrow stem cell-derived pluripotent cell (Muse cell).
  • ES embryonic stem
  • ntES nuclear transfer-mediated clone embryo-derived embryonic stem
  • GS cell germline stem cell
  • EG cell embryonic germ cell
  • iPS induced pluripotent stem
  • Muse cell fibroblast or bone marrow stem cell-derived pluripotent cell
  • An embryonic stem cell is a stem cell that has been established from an inner cell mass of an early embryo (e.g., a blastocyst) of a mammal such as a human and a mouse and that is pluripotent and can self-replicate.
  • the ES cell is an embryonic stem cell derived from an inner cell mass of a blastocyst, an embryo after an 8-cell stage and morula of a fertilized egg, and has an ability to differentiate into all the cells constituting an adult body, what is called pluripotency, and can self-replicate.
  • the ES cell was discovered in a mouse in 1981 ( M.J. Evans and M.H. Kaufman (1981), Nature 292: 154-156 ). Later, ES cell lines were established in primates such as a human and a monkey ( J. A. Thomson et al. (1998), Science 282: 1145-1147 ; J. A. Thomson et al. (1995), Proc. Natl. Acad. Sci.
  • An ES cell can be established by isolating an inner cell mass from a blastocyst of a fertilized egg of a target animal and culturing the inner cell mass on a fibroblast feeder layer.
  • the cells can be maintained by splitting the culture using a culture medium containing, for example, leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF).
  • LIF leukemia inhibitory factor
  • bFGF basic fibroblast growth factor
  • Human and monkey ES cells have been established and maintained in accordance with the procedures described in, for example, USP 5,843,780 ; Thomson JA, et al. (1995), Proc Natl. Acad. Sci. U S A. 92: 7844-7848 ; Thomson JA, et al. (1998), Science. 282: 1145-1147 ; H.
  • a culture medium for producing an ES cell it is possible to use DMEM/F-12 culture medium supplemented with, for example, 0.1 mM 2-mercaptoethanol, 0.1 mM non-essential amino acids, 2 mM L-glutamine, 20% KSR, and 4 ng/ml bFGF. Then, the human ES cell can be maintained at 37°C and 5% CO 2 under a humid atmosphere ( H. Suemori et al. (2006), Biochem. Biophys. Res. Commun., 345: 926-932 ). In addition, the ES cells should be divided every 3 to 4 days. At this time, the cells may be divided using, for example, 0.25% trypsin and 0.1 mg/ml collagenase IV in PBS containing 1 mM CaCl 2 and 20% KSR.
  • the ES cells can be selected, in general, by a real-time PCR method using, as an indicator, expression of gene markers such as alkaline phosphatase, Oct-3/4, and Nanog genes.
  • human ES cells can be selected by using, as an indicator, expression of gene markers such as OCT-3/4, NANOG, and ECAD genes ( E. Kroon et al. (2008), Nat. Biotechnol., 26: 443-452 ).
  • WA01 (H1) and WA09 (H9) are available from WiCell Research Institute and KhES-1, KhES-2, and KhES-3 are available from the Institute for Frontier Medical Sciences, Kyoto University (Kyoto, Japan).
  • the germline stem cell is a testis-derived pluripotent stem cell, which is a source of spermatogenesis.
  • This cell like the ES cell, can be induced to differentiate into various cell lineages, characterized in that when this cell is transplanted in a mouse blastocyst, for example, a chimeric mouse can be generated ( M. Kanatsu-Shinohara et al. (2003) Biol. Reprod., 69:612-616 ; K. Shinohara et al. (2004), Cell, 119: 1001-1012 ).
  • This cell can self-replicate in a culture medium containing glial cell line-derived neurotrophic factor (GDNF).
  • GDNF glial cell line-derived neurotrophic factor
  • the embryonic germ cell has been established from an embryonic primordial germ cell and, like an ES cell, is pluripotent. Also, an embryonic germ cell can be established by culturing a primordial germ cell in the presence of substances such as LIF, bFGF, and stem cell factor ( Y. Matsui et al. (1992), Cell, 70: 841-847 ; J. L. Resnick et al. (1992), Nature, 359: 550-551 ).
  • An induced pluripotent stem (iPS) cell can be created by introducing, as a form of DNA, RNA, or protein, specific reprogramming factors into a somatic cell, and is a somatic cell-derived induced stem cell that has substantially the same properties as of an ES cell, namely a cell having pluripotency and ability to self-replicate ( K. Takahashi and S. Yamanaka (2006) Cell, 126: 663-676 ; K. Takahashi et al. (2007), Cell, 131: 861-872 ; J. Yu et al. (2007), Science, 318: 1917-1920 ; Nakagawa, M. et al., Nat. Biotechnol.
  • iPS induced pluripotent stem
  • the reprogramming factors may be composed of genes that are specifically expressed in an ES cell, the products or non-coding RNAs thereof, genes that play a critical role in keeping an ES cell undifferentiated, the products or non-cording RNAs thereof, and/or low-molecular-weight compounds.
  • genes included in the reprogramming factors include Oct3/4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tel1, beta-catenin, Lin28b, Sall1, Sal14, Esrrb, Nr5a2, Tbx3 and Glis 1. These reprogramming factors may be used alone or in combination.
  • HDAC histone deacetylase
  • VPA valproic acid
  • trichostatin A sodium butyrate
  • MC 1293, M344 nucleic acid expression inhibitors
  • siRNAs and shRNAs against HDAC e.g., HDAC1 siRNA Smartpool (a registered trademark) (Millipore), HuSH 29mershRNA Constructs against HDAC1 (OriGene)
  • MEK inhibitors e.g., PD184352, PD98059, U0126, SL327, and PD0325901
  • glycogen synthase kinase-3 inhibitors e.g., Bio and CHIR99021
  • DNA methyltransferase inhibitors e.g., 5-azacytidine
  • histone methyltransferase inhibitors e.g., low
  • the reprogramming factors may be introduced into a somatic cell by using technology such as lipofection, fusion to a cell penetrating peptide (e.g., HIV-derived TAT and polyarginine), and microinjection in the form of protein.
  • a cell penetrating peptide e.g., HIV-derived TAT and polyarginine
  • examples of a technique used to introduce the reprogramming factors into a somatic cell include use of vectors (e.g., a virus, a plasmid, an artificial chromosome), lipofection, liposome, and microinjection.
  • vectors e.g., a virus, a plasmid, an artificial chromosome
  • lipofection liposome
  • microinjection examples of the virus vector include a retrovirus vector, a lentivirus vector (e.g., Cell, 126, pp.
  • the artificial chromosome vector examples include a human artificial chromosome (HAC), a yeast artificial chromosome (YAC), and a bacterial artificial chromosome (BAC, PAC).
  • HAC human artificial chromosome
  • YAC yeast artificial chromosome
  • BAC bacterial artificial chromosome
  • plasmid plasmids for mammalian cells may be used ( Science, 322:949-953, 2008 ).
  • the vector may contain a promoter, an enhancer, a ribosome binding sequence, a terminator, a poly-A site. Also, the vector, if needed, may further contain: a drug resistance gene (e.g., a kanamycin resistant gene, an ampicillin resistance gene, a puromycin resistance gene); a selection marker sequence (e.g., a thymidine kinase gene, a diphtheria toxin gene); and/or a reporter gene sequence (e.g., genes encoding green fluorescent protein, ⁇ -glucuronidase (GUS), FLAG).
  • a drug resistance gene e.g., a kanamycin resistant gene, an ampicillin resistance gene, a puromycin resistance gene
  • a selection marker sequence e.g., a thymidine kinase gene, a diphtheria toxin gene
  • a reporter gene sequence e.g., genes encoding green fluorescent protein, ⁇ -glucuronidas
  • the vector may contain LoxP motifs before and after the sequence.
  • RNA form In the case of an RNA form, a technique such as lipofection and microinjection may be used to introduce the reprogramming factors into a somatic cell. To reduce their degradation, RNA containing 5-methylcytidine and pseudouridine (TriLink Biotechnologies) may be used ( Warren L, (2010) Cell Stem Cell. 7: 618-630 ).
  • Examples of a culture medium for iPS cell induction include: 10 to 15% FBS-containing DMEM and DMEM/F12 or DME culture medium (these culture media optionally further containing LIF, penicillin/streptomycin, puromycin, L-glutamine, non-essential amino acids, ⁇ -mercaptoethanol, etc.); and commercially available culture media [e.g., mouse ES cell culture medium (TX-WES culture medium; Thromb-X N.V.), primate ES cell culture medium (primate ES/iPS cell culture medium; ReproCELL Inc.), serum-free media (mTeSR, Stemcell Technology, Inc.; Essential 8, Life Technologies, Inc.; StemFit, Ajinomoto, Inc.)].
  • FBS-containing DMEM and DMEM/F12 or DME culture medium these culture media optionally further containing LIF, penicillin/streptomycin, puromycin, L-glutamine, non-essential amino acids, ⁇ -mercapto
  • a culture protocol includes: causing reprogramming factors to contact somatic cells in 10% FBS-containing DMEM or DMEM/F12 culture medium at 37°C in the presence of 5% CO 2 and culturing the cells for about 4 to 7 days, and thereafter; plating the cells on a feeder cell (e.g., mitomycin C-treated STO cell, SNL cell, etc.) layer; and culturing, from about 10 days after the contact between the somatic cells and the reprogramming factors, the resulting cells in bFGF-containing primate ES cell culture medium, so that iPS-like colonies can be generated at about 30 to about 45 days or later after the contact.
  • a feeder cell e.g., mitomycin C-treated STO cell, SNL cell, etc.
  • the relevant cells are cultured on a feeder cell (e.g., mitomycin C-treated STO cell, SNL cell, etc.) layer in 10% FBS-containing DMEM culture medium (further optionally containing LIF, penicillin/streptomycin, puromycin, L-glutamine, non-essential amino acids, ⁇ -mercaptoethanol, etc.) at 37°C in the presence of 5% CO 2 , so that ES-like colonies can be generated after about 25 to about 30 days or later.
  • a feeder cell e.g., mitomycin C-treated STO cell, SNL cell, etc.
  • FBS-containing DMEM culture medium further optionally containing LIF, penicillin/streptomycin, puromycin, L-glutamine, non-essential amino acids, ⁇ -mercaptoethanol, etc.
  • somatic cells that are subject to reprogramming are used ( Takahashi K, et al. (2009), PLoS One.
  • an extracellular matrix e.g., Laminin-5 ( WO2009/123349 ), Matrigel (BD, Inc.), or iMatrix511 (Nippi, Inc.) is used.
  • an iPS cell may be established in a low oxygen condition (i.e., the level of oxygen of from 0.1% or more to 15% or less) ( Yoshida Y, et al. (2009), Cell Stem Cell. 5: 237-241 or WO2010/013845 )
  • the culture medium is changed to a fresh medium once a day from day 2 after initiation of the culture.
  • the number of somatic cells used for nuclear reprogramming has no limitation and ranges from about 5 ⁇ 10 3 to about 5 ⁇ 10 6 cells per 100 cm 2 culture dish.
  • the iPS cell can be selected in accordance with the morphology of a colony formed.
  • the iPS cell can be selected using, as indicators, genes (e.g., SSEA-1, SSEA-3, SSEA-4, TRA-2-54, TRA-1-60, and TRA-1-80) that are expressed when somatic cells are reprogrammed.
  • genes e.g., SSEA-1, SSEA-3, SSEA-4, TRA-2-54, TRA-1-60, and TRA-1-80
  • the term "somatic cell” refers to any animal cell (preferably, mammalian cells, including human cells) excluding reproductive cells (e.g., an egg cell, an oocyte, an ES cell) or totipotent cells.
  • reproductive cells e.g., an egg cell, an oocyte, an ES cell
  • totipotent cells e.g., an egg cell, an oocyte, an ES cell
  • somatic cell include, without limitation, any of fetal somatic cells, neonatal somatic cells, and mature, healthy or disease somatic cells. Additional examples include any of primary culture cells, subcultured cells, and cell lines.
  • tissue stem cells such as a neural stem cell, a hematopoietic stem cell, a mesenchymal stem cell, a dental pulp stem cell
  • tissue precursor cells such as a lymphocyte, an epithelial cell, an endothelial cell, a myocyte, a fibroblast (e.g., a skin cell, etc.), a hair cell, a hepatocyte, a gastric mucosa cell, an intestinal cell, a splenocyte, a pancreatic cell (e.g., a pancreatic exocrine cell, etc.), a brain cell, a pneumocyte, a nephrocyte, and an adipocyte.
  • tissue stem cells such as a neural stem cell, a hematopoietic stem cell, a mesenchymal stem cell, a dental pulp stem cell
  • tissue precursor cells such as a lymphocyte, an epithelial cell, an endothelial cell, a myocyte, a
  • somatic cells when used as a transplantable cell material, it is desirable from the viewpoint of there being no occurrence of rejection to use somatic cells having the same or substantially the same HLA genotype as of an individual that is subject to transplantation.
  • the term "substantially the same” means that the HLA genotype is matched to such an extent that the immune reaction against the transplanted cells can be suppressed by an immunosuppressive agent.
  • the somatic cells have an HLA genotype in which three gene loci: HLA-A, HLA-B, and HLA-DR are identical or four gene loci including an additional HLA-C are identical.
  • a nuclear transfer-mediated clone embryo-derived ES cell is an ES cell that is derived from a clone embryo generated using a nuclear transfer technique and has substantially the same properties as of a fertilized egg-derived ES cell ( T. Wakayama et al. (2001), Science, 292:740-743 ; S. Wakayama et al. (2005), Biol. Reprod., 72:932-936 ; J. Byrne et al. (2007), Nature, 450:497-502 ).
  • the nt ES (nuclear transfer ES) cell is an ES cell that has been established from an inner cell mass of a blastocyst of a clone embryo as obtained by switching the nucleus of an unfertilized egg to the nucleus of a somatic cell.
  • a nuclear transfer technique J.B. Cibelli et al. (1998), Nature Biotechnol., 16:642-646 ) and ES cell creation technology are combined ( Sayaka Wakayama (2008), "Jikken Igaku (Experimental Medicine)", vol. 26, no. 5 (special issue), pages 47-52 ).
  • the nucleus of a somatic cell is injected into an enucleated unfertilized egg of a mammal and the resulting egg is cultured for reprogramming for several hours.
  • the multilineage-differentiating stress enduring cell is a pluripotent stem cell as generated by the protocol described in WO2011/007900 . Specifically, fibroblasts or bone marrow stromal cells are treated with trypsin for an extended period and, preferably, the cells are treated for 8 or 16 h; and the cells are then subjected to suspension culture to yield pluripotent cells that are positive for SSEA-3 and CD105.
  • Cells sorted by the present invention may be administered pharmaceutically.
  • a therapeutic agent containing cells sorted by a method of the present invention When the cells sorted are endothelial cells, the cells may be administered for treatment of patients with severe ischemia including coronary artery disease and lower limb ischemia (e.g., Buerger disease, arteriosclerosis obliterans, etc.).
  • a vascular regeneration promoter containing endothelial cells sorted by the above-described method When the cells sorted are hepatocytes, the cells may be administered for treatment of liver failure such as chronic liver failure (e.g., hepatic cirrhosis) and acute liver failure.
  • hepatocytes obtained by the above-described method.
  • the cells sorted are insulin-producing cells
  • the cells may be administered for treatment of diabetes mellitus, in particular, type 1 diabetes.
  • a diabetes drug containing insulin-producing cells obtained by the above-described method.
  • the number of cells administered has no particular limitation as long as the dose can exert an advantageous effect on treatment.
  • the cells may be prepared by appropriately adjusting the number depending on the size of a lesion and/or the body size.
  • Table 7 lists PCR primers used below.
  • Primer name Sequence (5' -> 3') TagBFP_Tfwd TagBFP_Trev ACTCGAGATCTGTGCCCCAGTTTGCTAG pGEMTAP_MCS_Rev pGEMTAP_MCS_Fwd YF128_EXFP_Tfwd GAACCATGGGATCCGTGAGCAAGGGCGAGG YF129_EXFP_Trev TATGAGATCTCTTGTACAGCTCGTCCATG tagBFP fwd CACCGGTCGCCACCATGGGATCCAGCGAG TAPEGFP_IVTfwd CACCGGTCGCCACCATGGGATCCGTGAGCAAGGGC TAP_IVTrev TAP_T7_G3C fwd primer CAGTGAATTGTAATACGACTCACTATAGGGC Rev5UTR primer Fwd3UTR primer TCTAGACCTTCTGCGGGGC Rev3UTR2T20 3UTR120A rev
  • a fragment was amplified by PCR method using pTagBFP-Tubulin (evrogen) as a template and TagBFP_Tfwd and TagBFP_Trev as primers.
  • the resulting fragment was inserted into pGEM-TAP, a cloning vector originally constructed by the inventors.
  • the pGEM-TAP was constructed by PCR mutagenesis using pGEM-Teasy (Promega) as a template and pGFMTAP_MCS_Rev and pGEMTAP_MCS_Fwd as primers.
  • a fragment was amplified by PCR method using pEGFP-N1 (Clontech) as a template and YF128_EXFP_Tfwd and YF129_EXFP_Trev as primers.
  • the resulting fragment was inserted into pCM-TAP, a cloning vector originally constructed by the inventors.
  • the pCM-TAP was constructed by ligating a DraI-digested fragment of the above pGEM-TAP and a blunt-ended fragment of a NheI and Agel-digested pLysSRARE2 (Novagen).
  • the amplified PCR product was digested by DpnI (TOYOBO) at 37°C for 30 min, and was then purified using a MiniElute PCR purification kit (QIAGEN) in accordance with manufacturer's instructions (to yield what is called a tagBFP PCR product).
  • DpnI TOYOBO
  • QIAGEN MiniElute PCR purification kit
  • a fragment was amplified by PCR using the pCTp-EGFP as a template and TAPEGFP_IVTfwd and TAP_IVTrev as primers.
  • the PCR amplification was carried out and the resulting PCR product (called an EGFP PCR product) was purified.
  • a fragment was amplified by PCR using KOD-Plus-Neo (KOD-401, TOYOBO), the IVT_5prime_UTR, which is shown in Table 10, as a template, and TAP_T7_G3C fwd primer and Rev5UTR primer as primers in a solution of Table 9 as prepared in accordance with manufacturer's instructions.
  • the amplified PCR product was then purified using a MiniElute PCR purification kit (QIAGEN) in accordance with manufacturer's instructions (to yield what is called a 5'UTR PCR product).
  • QIAGEN MiniElute PCR purification kit
  • a fragment was amplified by PCR using KOD-Plus-Neo (KOD-401, TOYOBO), the above-described EGFP PCR product, 5' UTR PCR product and 3' UTR PCR product as templates, and TAP_T7_G3C fwd primer and 3UTR120A rev primer as primers in a solution of Table 11 as prepared in accordance with manufacturer's instructions.
  • the amplified PCR product was digested by DpnI (TOYOBO) at 37°C for 30 min, and was then purified using a MiniElute PCR purification kit (QIAGEN) in accordance with manufacturer's instructions.
  • IVT in vitro transcription
  • the IVT template for an miRNA switch was amplified by PCR using KOD-Plus-Neo (KOD-401, TOYOBO), the above-described tagBFP PCR product, each miRNA target 5'UTR oligo-DNA, and the above-described 3' UTR PCR product as templates, and T7FwdB primer and 3UTR120A rev primer as primers in a solution of Table 12 as prepared in accordance with manufacturer's instructions.
  • Table 13 lists each miRNA target 5' UTR oligo-DNA.
  • the amplified PCR product was digested by DpnI (TOYOBO) at 37°C for 30 min, and was then purified using a MiniElute PCR purification kit (QIAGEN) in accordance with manufacturer's instructions.
  • Each mRNA was produced using a MEGAscript T7 kit (Ambion) and each IVT template in accordance with the protocol described in Warren L, et al., Cell Stem Cell. 7(5): 618-30 (2010 ).
  • pseudouridine-5'-triphosphate and methylcytidine-5'-triphosphate (TriLink BioTechnologies) were used as alternatives for uridine triphosphate and cytidine triphosphate, respectively.
  • guanidine-5'-triphosphate was diluted five-fold with Anti-Reverse Cap Analog (New England Biolabs).
  • the reaction mixture was incubated at 37°C for 5 h, TURBO DNase (Ambion) was added, and the resulting mixture was then further incubated at 37°C for 30 min.
  • the resulting mRNA was purified through FavorPrep Blood/Cultured Cells total RNA extraction column (Favorgen Biotech), and was incubated with Antarctic phosphatase (New England Biolabs) at 37C° for 30 min. After that, the mRNA was further purified using an RNeasy Mini Elute Cleanup kit (QIAGEN).
  • Human umbilical vein endothelial cells (HUVECs) (C2517A) were purchased from Lonza, Inc., and were cultured using an EGM-2 Endothelial Cell Growth Medium-2 Bullet kit (Lonza).
  • the miRNA expression was profiled using Agilent Technologies Human miRNA Microarray Release 19.0 in accordance with the manufacturer's protocol. The data was analyzed using GeneSpring GX 12.6 software (Agilent Technologies).
  • human iPS cells used was 201B7 cell line described in Takahashi K, et al. Cell. 131: 861-72, 2007 , 409B2 (available from Riken BioResource Center), or 606A1 described in Okita K, et al., Stem Cells 31, 458-66, 2012 .
  • KhES1 was used and obtained from Kyoto University.
  • the human iPS cells or ES cells were treated with CTK solution (ReproCELL) for 2 min. Next, the solution was removed. Subsequently, the cells were treated with Accumax (Innovative Cell Technologies) for 5 min. Then, the cells were subjected to pipetting to single cells.
  • the resulting cells were recovered by centrifugation, transferred to a low-attachment 6-well dish (Corning), and cultured at 37°C under 5% oxygen condition in 1.5 ml/well of STEMPRO 34 (Invitrogen) supplemented with 2 mM L-glutamine, 150 ⁇ g/mL transferrin, 50 ⁇ g/mL ascorbic acid (sigma), 4 ⁇ 10 -4 M monothioglycerol (MTG), 0.5% penicillin/streptomycin (Invitrogen), 10 ⁇ M ROCK inhibitor (Y-27632), and 2 ng/mL BMP4 (R&D) to induce EB formation (day 0).
  • STEMPRO 34 Invitrogen
  • 2 mM L-glutamine 150 ⁇ g/mL transferrin, 50 ⁇ g/mL ascorbic acid (sigma), 4 ⁇ 10 -4 M monothioglycerol (MTG), 0.5% penicillin/streptomycin (Invitrogen), 10 ⁇ M ROCK inhibitor
  • IMDM Invitrogen
  • STEMPRO 34 supplemented with 2 mM L-glutamine, 150 ⁇ g/mL transferrin, 50 ⁇ g/mL ascorbic acid, 4 ⁇ 10 -4 M MTG, 10 ng/mL VEGF, and 1 ⁇ M IWP-3 (Stemgent).
  • the resulting culture was cultured at 37°C under 5% oxygen condition for 4 days.
  • the medium was changed to STEMPRO 34 supplemented with 2 mM L-glutamine, 150 ⁇ g/mL transferrin, 50 ⁇ g/mL ascorbic acid, 4 ⁇ 10 -4 M MTG, 0.5% penicillin/streptomycin, 10 ng/mL VEGF, and 5 ng/mL bFGF.
  • the resulting culture was cultured at 37°C under 5% oxygen condition for 4 days.
  • the medium change under the same conditions was performed once every two days.
  • Twelve days after the induction (day 12) the culture was transferred to an incubator with a normal oxygen level and was further cultured for 8 days. At this occasion, the medium change under the same conditions was performed once every two days.
  • the endothelial cells isolated were seeded at 2 ⁇ 10 5 cells/well on a Matrigel (BD)-coated 24-well plate, and were cultured in EGM-2 Bullet Kit medium. Next day, the culture was observed under a Biorevo BZ-9000 microscope.
  • BD Matrigel
  • hsa-miR-10b-5p, hsa-miR-126-3p, hsa-miR-126-5p, hsa-miR-196b-5p, and hsa-miR-216a-5p were used for microarray analysis; and top 5 miRNAs (hsa-miR-10b-5p, hsa-miR-126-3p, hsa-miR-126-5p, hsa-miR-196b-5p, and hsa-miR-216a-5p) specifically expressed in the endothelial cells were discovered ( Figure 1A ).
  • miRNA switches each having BFP and a sequence specifically recognized by each miRNA, (hereinafter, each referred to as miR-** miRNA switch) were constructed by the procedure described in Example 1 and each miRNA switch together with EGFP mRNA as a control was introduced into HUVECs.
  • miR-** miRNA switch each miRNA switch together with EGFP mRNA as a control was introduced into HUVECs.
  • HUVECs of interest were successfully isolated using each of miR-126-3p miRNA switch and miR-126-5p miRNA switch ( Figure 1B ).
  • both BFP mRNA and EGFP mRNA were together introduced into HUVECs. This result was compared with the above result and the isolation was verified.
  • the miR-126-3p miRNA switch and the miR-126-5p miRNA switch are useful in isolating endothelial cells from a heterogeneous cell population. Further, the cells that were isolated using the miR-126-5p miRNA switch were subjected to tube formation assay. The results demonstrated that the cells had angiogenic potential ( Figure 2C ).
  • Human primary hepatocytes were purchased from Bioreclamation IVT and were cultured in InVitroGRO CP Medium (Bioreclamation IVT) supplemented with Torpedo Antibiotic Mix (Bioreclamation IVT).
  • human iPS cells used was 201B6 cell line described in Takahashi K, et al., Cell. 131: 861-72, 2007 .
  • Hepatocytes were induced from human iPS cells in accordance with the method, with a modification, described in Kajiwara M, et al., Proc Natl Acad Sci U S A. 109: 12538-12543, 2012 .
  • the human iPS cells were seeded on a Matrigel-coated plate.
  • RPMI 1640 medium supplemented with 1 ⁇ B27 supplement, 100 ng/ml activin A, 10 ⁇ M Y-27632, and 1 ⁇ M CHIR99021.
  • the medium was changed to a culture medium in which Y-27632 had been removed from and 0.5 mM NaB had been added to the above medium.
  • the medium was changed to knockout DMEM supplemented with 20% knockout serum replacement (KSR), 1 mM L-glutamine, 1% nonessential amino acids, 0.1 mM 2-mercaptoethanol, 1% DMSO, 10 ng/ml FGF, and 20 ng/ml BMP4, and the cells were cultured for 6 days.
  • the medium was changed to hepatocyte culture medium (Lonza) supplemented with 20 ng/ml hepatocyte growth factor (HGF) and 20 ng/ml oncostatin M (OSM). The culture mixture was further cultured for seven days to yield hepatocytes.
  • HGF hepatocyte growth factor
  • OSM oncostatin M
  • hepatocytes To isolate hepatocytes, primary hepatocytes were used for microarray analysis; and top 5 miRNAs (hsa-miR-122-3p, hsa-miR-122-5p, hsa-miR-192-5p, hsa-miR-194-5p, and hsa-miR-215) specifically expressed in hepatocytes were discovered ( Figure 4A ).
  • Corresponding miRNA switches were constructed by the procedure of Example 1 and each miRNA switch together with EGFP mRNA as a control is introduced into hepatocytes derived from induced differentiation of human iPS cells. As a result, cells of interest were successfully isolated using miR-122-5p miRNA switch ( Figure 4B ).
  • human iPS cells used was 585A1 cell line described in Okita K, et al., Stem Cells 31, 458-66, 2012 .
  • Insulin-producing cells were induced from human iPS cells in accordance with the process described in Kunisada Y, et al., Stem Cell Res. 8:274-284, 2012 and Nakagawa, M, et al., Sci Rep 4, 3594, 2014 .
  • the cells were cultured for 1 day in RPMI 1640 medium supplemented with 1 ⁇ B27 supplement, 10 ⁇ M Y-27632, and 3 ⁇ M CHIR99021.
  • the medium was changed to RPMI 1640 medium containing 1 ⁇ B27 supplement, 100 ng/ml activin A, and 1 ⁇ M CHIR99021, and the cells were cultured for 3 days. Subsequently, the medium was changed to Improved MEM Zinc Option medium supplemented with 0.5 ⁇ B27 supplement, 1 ⁇ M dorsomorphin, 2 ⁇ M retinoic acid, and 10 ⁇ M SB431542, and the cells were cultured for 6 days.
  • the medium was changed to Improved MEM Zinc Option medium supplemented with 0.5 ⁇ B27 supplement, 10 ⁇ M forskolin, 10 ⁇ M dexamethasone, 5 ⁇ M Alk5 inhibitor II, and 10 mM nicotinamide, and the cells were cultured for 8 days.
  • miR-375 miRNA switch was constructed by the above procedure and the miR-375 miRNA switch together with EGFP mRNA as a control was introduced into insulin-producing cells derived from induced differentiation of human iPS cells. As a result, cells of interest were successfully isolated using the miRNA switch ( Figures 6A and 6B ). The cells isolated were confirmed to be positive for INSULIN by immunostaining ( Figure 6C ).

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Analytical Chemistry (AREA)
  • Plant Pathology (AREA)
  • Immunology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Vascular Medicine (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Claims (11)

  1. Procédé de tri d'un type de cellule tissulaire, comprenant l'introduction d'un ARNm sensible au miARN dans une population cellulaire sous la forme d'une molécule d'ARN, l'ARNm sensible au miARN comprenant :
    (i) une séquence d'acide nucléique spécifiquement reconnue par un miARN spécifiquement exprimé dans le type de cellule tissulaire à trier ; et
    (ii) une séquence d'acide nucléique correspondant à une région codante d'un premier gène marqueur ;
    (a) le type de cellule tissulaire à trier étant une cellule productrice d'insuline, un hépatocyte ou une cellule endothéliale ;
    (b) lorsque le type de cellule tissulaire à trier est une cellule productrice d'insuline, le miRNA spécifiquement exprimé dans la cellule étant le miR-375 ;
    (c) lorsque le type de cellule tissulaire à trier est un hépatocyte, le miRNA spécifiquement exprimé dans la cellule étant le miR-122-5P ; et
    (d) lorsque le type de cellule tissulaire à trier est une cellule endothéliale, le miRNA spécifiquement exprimé dans la cellule étant le miR-126-3P ou le miR-126-5P ;
    et une cellule ayant un faible niveau de traduction du premier gène marqueur étant classée comme cellule d'intérêt.
  2. Procédé selon la revendication 1, l'ARNm sensible au miRNA comprenant une séquence d'acide nucléique ayant (i) et (ii) liés dans une direction de 5' à 3'.
  3. Procédé selon la revendication 1 ou 2, le premier gène marqueur de (ii) étant au moins un gène choisi dans le groupe constitué par un gène codant pour une protéine fluorescente, un gène induisant l'apoptose et un gène suicide.
  4. Procédé selon la revendication 3, le gène codant pour la protéine fluorescente étant un gène codant pour la protéine fluorescente bleue (BFP).
  5. Procédé selon la revendication 3, le gène induisant l'apoptose étant un gène codant pour la protéine Bim.
  6. Procédé selon l'une quelconque des revendications 1 à 5, comprenant en outre l'introduction, dans la population cellulaire, d'un ARNm constitué d'une séquence comprenant un acide nucléique correspondant à une région codante d'un second gène marqueur, une cellule dans laquelle le second gène marqueur est traduit étant triée comme étant la cellule d'intérêt, et le second gène marqueur étant un gène fluorescent codant pour une protéine différente du premier gène marqueur, ou un gène de résistance aux médicaments.
  7. Procédé selon l'une quelconque des revendications 1 à 6, la population cellulaire étant une population de cellules qui a été induite à se différencier d'une cellule souche pluripotente.
  8. Procédé de production d'une type de cellules tissulaires, comprenant les étapes suivantes :
    (a) production, à partir d'une cellule souche pluripotente, d'une population de cellules contenant le type de cellules tissulaires ;
    (b) introduction, dans la population cellulaire de l'étape (a), d'un ARNm sensible au miRNA, constitué de séquences comprenant : (i) un acide nucléique reconnu spécifiquement par un miARN spécifiquement exprimé dans le type de cellules tissulaires à produire et (ii) un acide nucléique correspondant à une région codante d'un premier gène marqueur ; l'ARNm sensible au miARN étant sous la forme d'une molécule d'ARN ; et
    (c) tri d'une cellule ayant un faible niveau de traduction du premier gène marqueur de l'étape (b) ;
    (i) le type de cellules tissulaires à produire étant celui des cellules productrices d'insuline, des hépatocytes ou des cellules endothéliales ;
    (ii) lorsque le type de cellules tissulaires à produire est les cellules productrices d'insuline, le miRNA spécifiquement exprimé dans les cellules étant le miR-375 ;
    (iii) lorsque le type de cellules tissulaires à produire est des hépatocytes, le miRNA spécifiquement exprimé dans les cellules étant le miR-122-5P ; et
    (iv) lorsque le type de cellules tissulaires à produire est les cellules endothéliales, le miRNA spécifiquement exprimé dans les cellules étant miR-126-3P ou miR-126-5P.
  9. Procédé selon la revendication 8, l'ARNm sensible au miRNA comprenant une séquence d'acide nucléique ayant (i) et (ii) liés dans une direction de 5' à 3'.
  10. Procédé selon la revendication 8 ou 9, le premier gène marqueur de (ii) étant au moins un gène choisi dans le groupe constitué par un gène codant pour une protéine fluorescente, un gène induisant l'apoptose et un gène suicide.
  11. Procédé selon l'une quelconque des revendications 8 à 10, comprenant en outre l'introduction, dans la population cellulaire, d'un ARNm constitué d'une séquence comprenant un acide nucléique correspondant à une région codante d'un second gène marqueur, une cellule dans laquelle le second gène marqueur est traduit étant triée comme étant la cellule d'intérêt, et le second gène marqueur étant un gène fluorescent codant pour une protéine différente du premier gène marqueur, ou un gène de résistance aux médicaments.
EP16783256.7A 2015-04-22 2016-04-22 Méthode de tri de cellules tissulaires Active EP3318630B1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2015087805 2015-04-22
PCT/JP2016/062710 WO2016171235A1 (fr) 2015-04-22 2016-04-22 Méthode de tri de cellules tissulaires

Publications (3)

Publication Number Publication Date
EP3318630A1 EP3318630A1 (fr) 2018-05-09
EP3318630A4 EP3318630A4 (fr) 2019-04-24
EP3318630B1 true EP3318630B1 (fr) 2020-11-04

Family

ID=57143175

Family Applications (1)

Application Number Title Priority Date Filing Date
EP16783256.7A Active EP3318630B1 (fr) 2015-04-22 2016-04-22 Méthode de tri de cellules tissulaires

Country Status (4)

Country Link
US (2) US10501811B2 (fr)
EP (1) EP3318630B1 (fr)
JP (1) JP6960159B6 (fr)
WO (1) WO2016171235A1 (fr)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3121276B1 (fr) * 2014-03-20 2019-04-17 Kyoto University Procédé de tri de cardiomyocytes
WO2018165536A1 (fr) * 2017-03-10 2018-09-13 The Medical College Of Wisconsin, Inc. Thérapie génique modulée par riborégulateur pour maladies rétiniennes
US20210079440A1 (en) * 2017-07-12 2021-03-18 Kyoto University HIGH EXPRESSION mRNA SWITCH
WO2019031595A1 (fr) * 2017-08-10 2019-02-14 国立大学法人京都大学 Procédé de criblage de cellules rénales progénitrices

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2221371B1 (fr) 2007-11-22 2013-11-13 Japan Science and Technology Agency Système de régulation de la traduction dans une cellule ou dans un modèle cellulaire artificiel en utilisant de l'arn de faible masse moléculaire
US20110196017A1 (en) 2008-08-11 2011-08-11 Olson Eric N Micro-rna that promotes vascular integrity and uses thereof
BRPI1010873B1 (pt) 2009-04-30 2020-04-14 Fond Centro San Raffaele Del Monte Tabor usos de um vetor de genes, de uma composição farmacêutica e de uma célula
EP2580328A2 (fr) * 2010-06-11 2013-04-17 Cellartis AB Microarn pour la détection et l'isolement de types cellulaires cardiaques dérivés de cellules souches embryonnaires humaines
EP2749647B1 (fr) 2011-08-23 2018-07-04 National Institute of Biomedical Innovation Adénovirus apte à se répliquer conditionnellement
JP2016504050A (ja) * 2013-01-17 2016-02-12 モデルナ セラピューティクス インコーポレイテッドＭｏｄｅｒｎａ Ｔｈｅｒａｐｅｕｔｉｃｓ，Ｉｎｃ． 細胞表現型の改変のためのシグナルセンサーポリヌクレオチド

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
None *

Also Published As

Publication number Publication date
US20200056248A1 (en) 2020-02-20
JPWO2016171235A1 (ja) 2018-02-15
JP6960159B6 (ja) 2022-06-24
JP6960159B2 (ja) 2021-11-05
US10501811B2 (en) 2019-12-10
WO2016171235A1 (fr) 2016-10-27
EP3318630A1 (fr) 2018-05-09
US20180100203A1 (en) 2018-04-12
EP3318630A4 (fr) 2019-04-24

Similar Documents

Publication Publication Date Title
EP3042951B1 (fr) Nouveau procédé pour l'induction de cellules précurseurs neurales produisant de la dopamine
US20200056248A1 (en) Method for sorting tissue cells
JP6473077B2 (ja) 神経分化誘導用の多能性幹細胞
EP3121276B1 (fr) Procédé de tri de cardiomyocytes
JP6883791B2 (ja) 骨格筋前駆細胞の選択方法
EP2938721B1 (fr) Méthode d'induction des astrocytes
JP7236738B2 (ja) 神経前駆細胞の選別方法
US10689624B2 (en) Compound for identifying pluripotent cells
JP6877752B2 (ja) 細胞内在性タンパク質の識別方法
WO2022102742A1 (fr) Marqueur de surface cellulaire pour une purification à haut rendement de cellules de lignée de muscle squelettique et de cellules souches de muscle squelettique, et son utilisation
WO2021015086A1 (fr) Procédé de fabrication de cellules de myotube mâtures à partir de cellules souches du muscle squelettique
WO2020090836A1 (fr) Procédé de production de cellules

Legal Events

Date Code Title Description
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20171117

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

AX Request for extension of the european patent

Extension state: BA ME

DAV Request for validation of the european patent (deleted)
DAX Request for extension of the european patent (deleted)
RIC1 Information provided on ipc code assigned before grant

Ipc: C12N 5/16 20060101ALI20181203BHEP

Ipc: C12N 15/67 20060101ALI20181203BHEP

Ipc: C12N 15/113 20100101ALI20181203BHEP

Ipc: C12N 5/0735 20100101ALI20181203BHEP

Ipc: C12N 15/10 20060101AFI20181203BHEP

REG Reference to a national code

Ref country code: DE

Ref legal event code: R079

Ref document number: 602016047270

Country of ref document: DE

Free format text: PREVIOUS MAIN CLASS: C12N0015090000

Ipc: C12N0015100000

A4 Supplementary search report drawn up and despatched

Effective date: 20190325

RIC1 Information provided on ipc code assigned before grant

Ipc: C12N 15/10 20060101AFI20190319BHEP

Ipc: C12N 15/113 20100101ALI20190319BHEP

Ipc: C12N 5/0735 20100101ALI20190319BHEP

Ipc: C12N 5/16 20060101ALI20190319BHEP

Ipc: C12N 15/67 20060101ALI20190319BHEP

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: EXAMINATION IS IN PROGRESS

17Q First examination report despatched

Effective date: 20191108

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: GRANT OF PATENT IS INTENDED

INTG Intention to grant announced

Effective date: 20200604

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE PATENT HAS BEEN GRANTED

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: IPS ACADEMIA JAPAN, INC.

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

REG Reference to a national code

Ref country code: GB

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: CH

Ref legal event code: EP

REG Reference to a national code

Ref country code: AT

Ref legal event code: REF

Ref document number: 1330875

Country of ref document: AT

Kind code of ref document: T

Effective date: 20201115

REG Reference to a national code

Ref country code: IE

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: DE

Ref legal event code: R096

Ref document number: 602016047270

Country of ref document: DE

REG Reference to a national code

Ref country code: NL

Ref legal event code: MP

Effective date: 20201104

REG Reference to a national code

Ref country code: AT

Ref legal event code: MK05

Ref document number: 1330875

Country of ref document: AT

Kind code of ref document: T

Effective date: 20201104

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: GR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20210205

Ref country code: FI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20201104

Ref country code: NO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20210204

Ref country code: PT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20210304

Ref country code: RS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20201104

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20201104

Ref country code: BG

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20210204

Ref country code: IS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20210304

Ref country code: PL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20201104

Ref country code: LV

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20201104

Ref country code: ES

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20201104

Ref country code: AT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20201104

REG Reference to a national code

Ref country code: LT

Ref legal event code: MG9D

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: HR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20201104

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SM

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20201104

Ref country code: LT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20201104

Ref country code: EE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20201104

Ref country code: CZ

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20201104

Ref country code: RO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20201104

Ref country code: SK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20201104

REG Reference to a national code

Ref country code: DE

Ref legal event code: R097

Ref document number: 602016047270

Country of ref document: DE

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: DK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20201104

PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

26N No opposition filed

Effective date: 20210805

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20201104

Ref country code: AL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20201104

Ref country code: NL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20201104

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20201104

Ref country code: MC

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20201104

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20210422

REG Reference to a national code

Ref country code: BE

Ref legal event code: MM

Effective date: 20210430

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: CH

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20210430

Ref country code: LI

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20210430

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20210422

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20210304

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20210430

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: GB

Payment date: 20220425

Year of fee payment: 7

Ref country code: FR

Payment date: 20220425

Year of fee payment: 7

Ref country code: DE

Payment date: 20220429

Year of fee payment: 7

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: CY

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20201104

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: HU

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT; INVALID AB INITIO

Effective date: 20160422

REG Reference to a national code

Ref country code: DE

Ref legal event code: R119

Ref document number: 602016047270

Country of ref document: DE

GBPC Gb: european patent ceased through non-payment of renewal fee

Effective date: 20230422

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: GB

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230422

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: GB

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230422

Ref country code: FR

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20230430

Ref country code: DE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20231103

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20201104